STRUCTURES AND MECHANISMS OF CITRATE ENZYMES

柠檬酸酶的结构和机制

• 批准号: 3224776
• 负责人: PAUL A SRERE
• 金额: $ 13.27万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224776
• 关键词: Escherichia coli; X ray crystallography; chemical structure function; citrate synthase; complementary DNA; conformation; enzyme mechanism; enzyme structure; genetic manipulation; molecular cloning; molecular site; point mutation; protein engineering; protein sequence; radiotracer; structural genes; swine

The goal of the research is to examine the structure and function of citrate synthase (CS) from pig and E. coli by using site-directed mutagenesis. Citrate synthase is an excellent choice to examine enzyme structure and function by using such molecular biological techniques. It is a key enzyme in aerobic energy production and metabolite interconversions. It is an important example of stereospecificity in enzyme reactions, and two distinct enzyme conformations participate during catalysis. In addition, pig citrate synthase (PCS) and E. coli citrate synthase (ECCS) have been crystallized and the three dimensional structure of PCS determined. Therefore, CS is an attractive enzyme to study by site-directed mutagenesis because of the possibility of obtaining the DNA, the protein crystals, and an in depth mechanism for the mammalian and bacterial forms of the enzyme. This additional comparative aspect between the CS from a eucaryote and a procaryote will enhance our ability to choose sites for mutagenesis, and also will explain the two different reaction mechanisms, protein structures, and regulatory behaviors of these divergently related proteins. To define in detail the reaction mechanism, structure, and biological function of CS, the cDNA encoding PCS will be isolated and sequenced. Site-directed mutagenesis of the PCS and ECCS DNAs will used to alter codons for catalytic and structural amino acid residues. The mutated and control DNAs will be expressed in vitro, and the synthesized proteins will be purified and crystallized. The partial enzyme reactions catalyzed by the control and mutated PCS and ECCS proteins will be determined and compared. The three dimensional structures of the control and mutated PCS and ECCS proteins will be compared to the known X-ray structure of PCS. In addition, the gene for an E. coli mutant that codes for the dimeric (eucaryotic) form of the enzyme will be isolated and sequenced. The amino acid sequence for the mutant ECCS will be compared to the PCS and non-mutant ECCS and may identify particualr amino acid residues that are important to subunit assembly or enzymatic function.

这项研究的目的是检查结构和功能 利用定点技术从猪和大肠杆菌中分离柠檬酸合成酶 诱变。柠檬酸合成酶是一个很好的检查选择 利用这种分子生物学研究酶的结构和功能 技巧。它是有氧能量产生和释放的关键酶 代谢物相互转化。这是一个重要的例子 酶反应中的立体特异性，以及两种不同的酶 构象参与催化过程。此外，猪 柠檬酸合成酶(PCS)和大肠杆菌柠檬酸合成酶(ECCS) 结晶和PCS的三维结构 下定决心。因此，CS是一种很有吸引力的研究酶 定点突变是因为有可能获得 DNA，蛋白质晶体，以及一种深度机制 哺乳动物和细菌形式的酶。这是额外的 真核生物的CS和A的CS的比较方面 原核生物将增强我们选择地点的能力 诱变，也将解释两种不同的反应 它们的机制、蛋白质结构和调节行为 不同的相关蛋白质。要详细定义反应 CS的作用机制、结构和生物学功能 编码PCS将被分离和排序。站点定向 PCS和ECCS DNA的突变将用于改变密码子 用于催化和结构氨基酸残基。突变的和 对照DNA将在体外表达，合成的 蛋白质将被提纯和结晶。部分酶 对照和突变的PCS和ECCS催化的反应 蛋白质将被测定和比较。三度空间 对照和突变的PCS和ECCS蛋白的结构将 与已知的PCS的X射线结构相比较。此外, 编码二聚体的大肠杆菌突变体的基因 (真核)形式的酶将被分离和测序。 将比较突变的eccs的氨基酸序列。 与PCS和非突变ECC结合，并可识别特定的 氨基酸残基对亚基组装或 酶功能。